title: Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein.
abstract: cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits from Yarrowia lipolytica are encoded by single genes, TPK1 and RKA1, respectively. Here we performed the heterologous expression, purification and characterization of the R subunit from Y. lipolytica yeast cells, and explored the main biochemical features of the PKA. The purified recombinant R, active and capable to interact with C subunit was used to prepare highly specific polyclonal antiserum. Sucrose-gradient centrifugation and gel filtration analysis of both recombinant and native R revealed the monomeric nature of this subunit. Hydrodynamic parameters of the holoenzyme indicated that Y. lipolytica PKA is a dimer of 90 kDa composed of an R subunit of 42 kDa and a C subunit of 39 kDa. The identification of the N-terminal sequence was carried out by mass spectrometry analysis of the purified native R subunit. The differences between N-terminal sequences of R subunits from Y. lipolytica and other organisms, particularly a short linker that spans the inhibitory site, were discussed as the possible cause of the lack of dimerization. R was identified as a type II subunit since our results indicated that it was phosphorylated in vivo by C at S124 identified by anti-phospho-PKA substrate (RRXS/T) antibody.
authors.affiliation: Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, INBA-CONICET, Avda. San Martin 4453, C1417DSE Buenos Aires, Argentina.
authors.forename: Florencia
authors.initials: F
authors.lastname: Kronberg
authors.forename: Romina
authors.initials: R
authors.lastname: Giacometti
authors.forename: José
authors.initials: J
authors.lastname: Ruiz-Herrera
authors.forename: Susana
authors.initials: S
authors.lastname: Passeron
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057446:Hydrodynamics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025062:Yarrowia / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D020035:Holoenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases